Metastasis remains the major cause of therapeutic failure, poor prognosis and high mortality in breast and prostate cancer patients treated with surgery or radiotherapy. Novel approaches to reduce metastatic incidences and improve overall survival of cancer patients clearly are needed. The current proposal is focused on therapeutically targeting a key player in the metastatic cascade; the cysteine protease cathepsin L (CTSL). CTSL is over-expressed and hyper-activated in primary tumors and metastatic lesions in many cancer types, including breast and prostate cancer. Its over-activation correlates with tumor invasiveness and tumor grade, yielding a poor patient prognosis. CTSL initiates migratory and invasive processes through direct degradation of E-Cadherin, extracellular matrix, and basement membrane components. Cathepsins also critically contribute to the process by which metastatic tumor cells in the skeleton stimulate osteoclast-mediated bone resorption; a source of significant morbidity in prostate and breast cancer patients. CTSL may therefore provide a unique target in the metastatic process. The goal of the present proposal is to explore the anti-metastatic potential of CTSL inhibition using a small molecule targeting approach in human (MBA-MB-231, PC-3ML) and rodent (TRAMP) breast and prostate cancer models. Aim 1 will assess the impact of selective small molecule CTSL inhibitors on key functional steps associated with tumor cell dissemination including ECM degradation, cell motility, and cell invasion. In Aim 2 the effect of hypoxic and acidic tumor microenvironments, as commonly occur in solid tumors, on CTSL release, activity, and response to small molecule inhibition will be assessed. Aim 3 will examine whether cathepsin targeting can inhibit bone resorption; a key process leading to skeletal morbidity in cancer patients. Aim 4 will evaluate the in vivo anti-metastatic potential of CTSL targeting using both molecular suppression and selective therapeutic inhibition of CTSL. First, a known number of MDA-MB- 231 or PC-3ML cells will be injected via the intracardiac route and the efficacy of CTSL inhibitors (KGP94 or KGP207) will be determined by (i) non-invasive monitoring of the metastatic burden using an in vivo imaging system, and (ii) quantifying the number of bone metastases. Second, select studies will evaluate the effect of CTSL interference on the spread of cancer cells from primary tumors to secondary sites in (i) mice undergoing surgical removal of orthotopically transplanted breast tumors and (ii) the spontaneous prostate cancer metastasis TRAMP model. Finally, Aim 5 will address the role of CTSL and its inhibition to tumor induced angiogenesis and vasculogenesis, key contributors to metastasis. The successful completion of the proposed studies will provide evidence that CTSL is a potential therapeutic target for novel anti-metastatic cancer therapies. Preclinical investigations of approaches targeting the spread of cancer cells may ultimately provide clinical benefit by enabling the development of future treatment regimes designed to enhance outcomes of existing surgical interventions and radiotherapy treatments for advanced breast and prostate cancer patients.